Targeted gene deletions for polysaccharide slime formers

ABSTRACT

The present invention provides improved diutan polysaccharides produced by mutant  Sphingomonas  strains containing at least one genetic modification that favors slime-forming diutan production that exhibits increased viscosity. Methods of making the mutant  Sphingomonas  strains and the culture broth containing such mutant  Sphingomonas  strains are also provided.

This application claims the benefit of U.S. Provisional Application No.60/649,559, filed Feb. 4, 2005.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of sphingan polysaccharideproduction. In particular, it relates to site-directed genetic methodsfor improving sphingan-producing strains.

BACKGROUND OF THE INVENTION

Sphingomonas strains, such as ATCC 53159 and ATCC 31461, produce copiousamounts of capsular polysaccharide. While under some conditionspolysaccharide may be released from the cell [5, 6], during growth withabundant carbon source as in fermentation, the polysaccharide is firmlyattached to the cell surface. Attempts to increase productivity offermentations for diutan and gellan may be limited by the capsularnature of the polysaccharide, which may impair uptake of nutrients.Also, if there are a limited number of sites for biosynthesis of thepolysaccharide, there may be a maximum amount of polysaccharide that canbe produced by each cell. The polysaccharide gellan has been observed tobe involved in cell clumping since mutants that do not make anypolysaccharide grow uniformly in suspension [3]. These cell clumps mayinterfere with techniques such as determination of cell number byoptical density, centrifugation of cells, e.g., for isolation of DNA orprotein, and separation or lysis of cells for polysaccharidepurification.

The mechanism of attachment and the genes involved in attachment ofpolysaccharide to the cell surface in Sphingomonads have not beenpreviously determined. Induced mutants of Sphingomonas strains ATCC31461, ATCC 31555, ATCC 31554, and ATCC 21423 that producepolysaccharide in a slime form have been isolated, but the genes mutatedwere not determined, and the methods of inducing and selecting themutants were not disclosed [10]. Genes for biosynthesis of gellan [3,8], diutan [1] and sphingan S-88 [9] have been isolated. The functionsof many of these genes were assigned by biochemical tests or by homologyto genes of known functions in databases such as GenBank. For example,genes have been identified that are involved in assembly of thetetrasaccharide repeat unit [7, 8], and in synthesis of the precursordTDP-L-rhamnose [3, 9]. It would be expected that genes affecting onlyattachment of polysaccharide to the cell surface would still have thepolysaccharide producing phenotype (i.e., mucoid colonies on solid mediaand viscous broth).

A cluster of 18 genes for gellan biosynthesis spanning 21 kb wasdescribed, in addition to four genes for gellan synthesis not in thecluster Harding N. E., Y. N. Patel, and R. Coleman, 2004. Organizationof genes required for gellan polysaccharide biosynthesis in Sphingomonaselodea ATCC 31461. J Ind Microbiol Biotech 31:70-82, reference 3. TheDNA sequences were deposited in GenBank in June 2003 (Accession numberAY217008). Among the genes in the cluster were gelM, gelN, and gelI. Adeletion of most of adjacent genes gelM and gelN was constructed. ThegelI gene was inactivated by an insertion. The gelM-gelN deletion strainand the gelI mutant were shown to produce somewhat reduced amounts ofgellan and more fluid broths, and the gellan produced was shown to havethe same composition as gellan from the wild-type strain. The attachmentof the polysaccharide to the cell was not reported

The Sphingomonas elodea gelR, gelS, and gelG genes appear to be in anoperon in the same order as in the S-88 sps gene cluster, but notadjacent to the genes in the cluster of 18 genes (reference 3). The GelRprotein was somewhat smaller than its S-88 homolog (659 vs. 670 aminoacids) with 49% identity, and had homology to surface layer proteins andother membrane proteins. The DNA sequences of gelR, gelS and gelG geneswere deposited in GenBank in June 2003 (Accession number AY220099). Nomutation in gelR was constructed in this report (reference 3). Yamazakiet al. report that strains with mutations in gene spsR were stillmucoid, indicating that they produce polysaccharide, but thepolysaccharide was not characterized as to rheology or attachment to thecell ([9] and T. J. Pollock et al. DNA segments and methods forincreasing polysaccharide production. U.S. Pat. No. 5,854,034.

Yamazaki described classical mutants of four Sphingomonas strains thatproduce polysaccharide as slime rather than attached to the cell. U.S.Pat. No. 6,605,461. Yamazaki did not describe how to screen mutagenizedcultures for the slime phenotype. Yamazaki did not identify which geneor genes were mutated.

Sa-Correia reviewed work done on isolation of genes for gellansynthesis. Sa-Correia I., A. M. Fialho, P. Videira, L. M. Moreira, A. R.Marques and H. Albano. 2002. Gellan gum biosynthesis in Sphingomonaspaucimobilis ATCC 31461: Genes, enzymes and exopolysaccharide productionengineering. J Ind Microbiol Biotechnol. 29: 170-176. Sa-Correiadescribed partial sequencing of some genes including urf32 and urf26(=gelM and gelN of reference 3 above). The complete sequences of thesegenes were deposited in GenBank in April 2003 (GenBank Accession numberAY242074). No function of these genes is reported. In the GenBanksubmission, genes urf32 and urf26 were merely designated as putativemembrane protein and putative exported protein, respectively. Nosequence for gelI or gelR was deposited.

Coleman describes the isolation of genes for diutan biosynthesis andinvestigation of some gene functions. R. Coleman, 2001, Cloning andanalysis of Sphingomonas sp. ATCC 53159 polysaccharide genes. Master'sThesis, San Diego State University. The dpsM and dpsN genes, which weredesignated by Coleman as orf3 and orf4, were described but functionswere not indicated.

A cluster of genes for biosynthesis of the S-88 polysaccharide fromSphingomonas strain ATCC 31554 was described. Yamazaki M., L. Thorne, M.Mikolajczak, R. W. Armentrout and T. J. Pollock. 1996. Linkage of genesessential for synthesis of a polysaccharide capsule in Sphingomonasstrain S88. J Bacteriol 178: 2676-2687 and U.S. Pat. No. 5,854,034. Thefunctions of genes urf32 and urf26 (homologs of dpsM, gelM and dpsN,gelN), and spsI (homolog of gelI, dpsI) were not described. Gene spsR(homolog of gelR, dpsR) was described as encoding a protein remotelysimilar to bacterial and fungal polysaccharide lyases The DNA sequenceswere deposited in GenBank (Accession number U51197).

There is a continuing need in the art to improve methods of makingindustrially useful sphingans and the properties of the sphingans.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method is provided ofmaking a bacterium. The bacterium is of the genus Sphingomonas andcomprises a mutation in one or more genes selected from the groupconsisting of genes M, N I or R of the sphingan polysaccharidebiosynthetic gene cluster. The M and N genes are also referred to insome publications, e.g., references 8 and 9, as genes urf32, urf26,respectively (for unknown reading frame). A segment of genomic DNA of afirst bacterium of the genus Sphingomonas is isolated. The segmentcomprises all or part of genes M and/or N or I or R of the sphinganpolysaccharide biosynthetic gene cluster. A mutation in the segment isinduced to form a mutated segment. The mutated segment is introducedinto a second bacterium of the genus Sphingomonas. The second bacteriumcomprises wild-type genes M and/or N, or I, or R of the sphinganpolysaccharide biosynthetic gene cluster. A progeny of the secondbacterium in which the mutated segment has integrated in the genome andreplaced wild-type genes M and/or N or I, or R of the sphinganpolysaccharide biosynthetic gene cluster of the second bacterium isisolated. The Sphingomonas bacterium may or may not be S. elodea.

According to another embodiment of the invention another method isprovided of making a bacterium of the genus Sphingomonas which comprisesa mutation in one or more genes selected from the group consisting ofgenes M, N, I, and R of the sphingan polysaccharide biosynthetic genecluster. Two non-contiguous segments of genomic DNA of a first bacteriumof the genus Sphingomonas are isolated. The segments flank or includegenes M and N of the sphingan polysaccharide biosynthetic gene cluster.Similarly, segments flanking gene I or gene R can be isolated. The twonon-contiguous segments are ligated together. The ligated non-contiguoussegments are introduced into a second bacterium of the genusSphingomonas. The second bacterium comprises wild-type genes M and/or Nor I or R of a sphingan polysaccharide biosynthetic gene cluster. Aprogeny of the second bacterium in which the ligated segment hasintegrated in the genome and replaced wild-type genes M and/or N or I orR of a sphingan polysaccharide biosynthetic gene cluster of the secondbacterium is isolated. The Sphingomonas bacterium may or may not be S.elodea.

According to yet another embodiment of the invention a composition isprovided. The composition comprises a native gellan polysaccharide withgel strength greater than that of an equivalent weight of native gellanfrom a capsular strain.

According to yet another embodiment of the invention a composition isprovided. The composition comprises a diutan polysaccharide whichimparts to a fluid an increased viscosity relative to an equivalentweight of diutan produced by strain ATCC 53159.

According to another embodiment of the invention an isolated andpurified bacterium of the genus Sphingomonas is provided. The bacteriumcomprises a deletion in one or more genes selected from the groupconsisting of genes M, N, I, and R of the sphingan polysaccharidebiosynthetic gene cluster. The bacterium can be cultured in a culturemedium under conditions suitable for producing sphingan polysaccharideto produce sphingan polysaccharide in the culture medium. The culturebroth of the bacterium can be used directly as a viscosifier or gellingagent, or after precipitation with alcohol. Alternatively, the culturebroth can be subjected to a procedure to remove bacteria from theculture broth prior to use as a viscosifier or gelling agent or recoveryfrom the broth. The Sphingomonas bacterium may or may not be S. elodea.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with new methods,strains, and compositions for making viscosifiers and gelling agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of gene clusters for polysaccharide biosynthesis inSphingomonas strains ATCC 31554, ATCC 31461 and ATCC 53159.

FIG. 2. Slime forming characteristics of S60WTC gelM-gelN mutants

FIG. 3. Slime forming characteristics of S60WTC gelN and gelI Mutants

FIG. 4. Sequence of DNA at the site of deletion of dpsN (SEQ ID NO: 19),and amino acid sequence of the fusion peptide (SEQ ID NO: 20).

FIG. 5A-5C. Slime forming characteristics of dpsN mutants

FIG. 6A-6B. Slime forming characteristics of dpsM mutants

DETAILED DESCRIPTION OF THE INVENTION

Genes have been identified that control the attachment of polysaccharideto bacterial cells in two Sphingomonas strains. Deletion of either oneor both genes gelM (dpsM) and gelN (dpsN) or inactivation of gelIresults in polysaccharide being released into the medium as slime ratherthan attached to the cell surface as capsular polysaccharide. Formationof slime form of polysaccharide eases handling of bacterial cultures,improves mixing during fermentation, may increase production, and insome cases improves rheology of the polysaccharide. Site directedmutagenesis is advantageous over random mutagenesis and screening forslime-forming mutants for many reasons, including speed and avoidance ofunrelated mutations. Inactivation of the gene gelR was found to improvethe rheology (gel strength) of the slime form of gellan polysaccharide.

Orthologs of dpsM, dpsN, gelM, gelN, and gelI can be inactivated in anySphingomonas strain to obtain the slime-forming phenotype. Orthologs ofgelR can be inactivated to prevent degradation of the polysaccharideresulting in improved rheology. Suitable Sphingomonads include withoutlimitation those which make rhamsan (ATCC 31961), welan (ATCC 31555),gellan (ATCC 31461), and diutan (ATCC 53159) and strains makingpolysaccharides S7 (ATCC 21423), S88 (ATCC 31554), S198 (ATCC 31853) andNW11 (ATCC 53272). The ATCC numbers refer to the deposit numbers of thestrains at the American Type Culture Collection. These are exemplifiedby S. elodea ATCC 31461 and Sphingomonas sp. ATCC 53159, but otherstrains can be used. Suitable Sphingomonads which can be used includeSphingomonas adhaesiva, Sphingomonas aerolata, Sphingomonas alaskensis,Sphingomonas aquatilis, Sphingomonas aromaticivorans, Sphingomonasasaccharolytica, Sphingomonas aurantiaca, Sphingomonas capsulata,Sphingomonas chlorophenolica, Sphingomonas chungbukensis, Sphingomonascloacae, Sphingomonas echinoides, Sphingomonas elodea, Sphingomonasfaeni, Sphingomonas herbicidovorans, Sphingomonas koreensis,Sphingomonas macrogoltabidus, Sphingomonas mali, Sphingomonas melonis,Sphingomonas natatoria, Sphingomonas parapaucimobilis, Sphingomonaspaucimobilis, Sphingomonas pituitosa, Sphingomonas pruni, Sphingomonasrosa, Sphingomonas sanguinis, Sphingomonas sp., Sphingomonas stygia,Sphingomonas subarctica, Sphingomonas suberifaciens, Sphingomonassubterranea, Sphingomonas taejonensis, Sphingomonas terrae, Sphingomonastrueperi, Sphingomonas ursincola, Sphingomonas wittichii, Sphingomonasxenophaga, Sphingomonas yabuuchiae, and Sphingomonas yanoikuyae.Orthologs can be identified on the basis of gene location andorganization in a sphingan biosynthetic gene cluster, on the basis ofoverall homology, and/or on the basis of domain homology. Typically thelevel of overall homology will be greater than 44%, often greater than55%, 66%, 77%, 88%, or 98% with one of the dpsM, dpsN, gelM, gelN, gelIor gelR genes. An ortholog desirably has homology greater than 80% withat least one of these four genes.

Site directed mutagenesis can be used to make mutations in a desiredknown target gene or genomic region. This eliminates the trial-and-errornature of random induced mutagenesis or spontaneous mutagenesis.Formation of deletions insures that the mutations will not revert, as ispossible with point (substitution) mutations and insertion mutations,for example. Deletions also have the benefit of not employing exogenousDNA, such as drug resistance markers or other environmentallyundesirable markers.

An isolated segment of genomic DNA comprising the M and/or N, I or R ofthe sphingan biosynthetic gene cluster or flanking DNA is DNA that isnot connected to genomic DNA to which it is normally attached. IsolatedDNA can be obtained by purification from natural sources, by synthesis,or by amplification, as non-limiting examples. The isolated DNA willtypically be on a fragment of DNA in vitro, but isolated DNA could alsobe on a vector, such as a plasmid or transducing phage, which containsthe desired portion of the Sphingomonas genome. Flanking DNA istypically from the genomic regions immediately adjacent to the M and/orN, I or R within about 500 bp of the genes, or within about 1-2 kb ofthe genes.

Any method known in the art can be used to introduce a mutation into anisolated segment comprising all or part of genes M and/or N, I or R ofthe sphingan biosynthetic gene cluster. A deletion can be introducedusing restriction endonucleases, for example, and rejoining formerlynon-contiguous nucleotides. A deletion can be formed by amplifying andjoining two non-contiguous segments of the genes or two non-contiguoussegments of DNA flanking the target gene. An insertion can be made in anisolated segment using endonuclease digestion and ligation. Chemicalmutagenesis can be used on an isolated segment of genomic DNA. Anymutagenesis method can be selected and used according to the particularcircumstances.

After mutations have been induced, the segment of genomic DNA can bereintroduced into a recipient bacterium. Typically, but not necessarily,the recipient will be of the same species as the donor of the segment.Any method known in the art for introducing exogenous DNA into abacterium can be used. Suitable methods include without limitationelectroporation, conjugation, spheroplast formation, calcium chlorideprecipitation and transformation, liposomes, and viral transduction. Anynucleic acid introduction method can be selected and used according tothe particular circumstances.

If the segment of mutated genomic DNA introduced into the recipientbacterium does not have a means of replicating itself, then it mustintegrate into a replicon in the recipient bacterium in order to bemaintained. Typically such an integration event will integrate theentire incoming plasmid. One can detect a marker on the introduced DNAto identify that the DNA has integrated. In order to detect resolutionof the integrate, one can screen or select for loss of a marker on theintroduced DNA. Suitable markers for accomplishing this are known in theart, and any can be used as the circumstances dictate. To determine theisolates in which the introduced version of the sphingan genes replacesthe wild-type version in the recipient, the size or sequence of the DNAcan be determined, for example, by PCR.

As demonstrated below, the slime form of sphingan produced for exampleby a sphingan biosynthetic gene cluster gene M and/or N, mutant may haveimproved rheological properties over the form which is attached tobacterial cells. Such improved Theological properties are reflected inthe ability of the same weight of material to provide more viscosifyingpower. Such improvement may be modest, such as at least 5% 10%, 15%, 20%or 25%, or it can be more substantial, with an improvement of at least30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to the sphingan producedby the capsule-forming parent. Rheologically properties can be measuredusing any technique which is known in the art. Suitable techniquesinclude without limitation the measurement of Low Shear Rate Viscosity(“LSRV”) in tap water solutions and the measurement of Sea WaterViscosity (“SWV”) in high salt solutions.

The slime form of gellan, produced for example by a gelN mutant incombination with a mutation in the putative lyase gene gelR results information of gellan of high gel strength. The gel strength willtypically e greater than 1000, whereas the capsular strain typicallyproduces a gellan with gel strength of 700-900 but less than 1000.

Purified bacteria according to the present invention are those whichhave been microbiologically purified, for example using liquid dilutiontechniques or streaking on solid media to form single colonies. Standardtechniques known in the art of microbiology can be used for thispurpose.

Mutants according to the present invention can be cultured and grownusing the same or similar techniques as used for the parental strains.Liquid culture properties of the mutants may be improved, permittingincreased aeration and mixing. The culture broth of the mutant may alsoprovide more efficient recovery than with the attached form ofpolysaccharide. In addition, the mutants may also provide a product withimproved clarity relative to the attached form of polysaccharide.Bacteria may optionally be removed from the polysaccharide produced bythe mutant by filtration, centrifugation, or by sedimentation. Theculture broth can be chemically, enzymatically, or thermally (hot orcold) treated before or after bacteria removal, as desired.

The genes from S. elodea ATCC 31461 involved in gellan attachment to thecell surface are gelM and gelN. (FIG. 1; SEQ ID NO: 13) and gelI (FIG.1, SEQ ID NO: 25). A strain has been constructed that has a deletion ofmost of genes gelM and gelN, resulting in the slime-forming phenotype. Aspecific deletion of gelN has also been constructed, and an insertion ingene gelI. Both of these mutations result in the slime-formingphenotype. The coding sequences of gelM and gelN are at nucleotides501-1382 and 1366-2064, respectively, in SEQ ID NO: 13. The encodedamino acid sequences are shown at SEQ ID NOs: 16 and 15, respectively.The coding sequences of gelI is at nucleotides 501 to 1403,respectively, in SEQ ID NO: 25. The encoded amino acid sequences areshown at SEQ ID NOs: 26 A deletion of gene gelR was found to result inimproved gel strength for gellan in the slime form. The coding sequencesof gelR is at nucleotides 478 to 2457, respectively, in SEQ ID NO: 27The encoded amino acid sequences are shown at SEQ ID NOs: 28. The genesfrom Sphingomonas sp. ATCC 53159 involved in diutan attachment to thecell surface are dpsM and dpsN (FIG. 1; SEQ ID NO: 14), and presumablydpsI based on homology to gelI. Deletions of each of genes dpsM and dpsNhave been constructed and both result in the slime-forming phenotype.The coding sequences of dpsM and dpsN are at nucleotides 456-1337 and1321-2019, respectively, in SEQ ID NO: 14. The encoded amino acidsequences are shown at SEQ ID NOs: 18 and 17, respectively.

It will be apparent to those skilled in the art that the same or similarmethods used for gellan synthesis may also be used for diutan synthesis.Thus mutations in genes dpsI and dpsR could readily be constructed Thecoding sequences of dpsI is at nucleotides 501-1472, respectively, inSEQ ID NO: 29 The encoded amino acid sequences are shown at SEQ ID NOs:30 The coding sequences of dpsR is at nucleotides 501-2498,respectively, in SEQ ID NO: 31. The encoded amino acid sequences areshown at SEQ ID NOs. 32.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Production of Gellan Slime-Forming Mutants

For construction of mutants of Sphingomonas elodea a derivative of ATCC31461 designated S60 wtc¹ was used, which has improved uptake of DNA.This strain can be readily made by one skilled in the art. PCRamplification was used to amplify regions flanking the gelM-gelN genes.See reference 3. The amplified fragments were cloned into the pLO2vector and introduced into S60 wtc by conjugation to replace the gelMand gelN genes on the genome with the deletion, by double crossoverhomologous recombination. Vector pLO2 does not replicate in S60 wtc, soinitial selection for kanamycin resistance selects for those colonies inwhich the plasmid has integrated into the chromosome by homologousrecombination. The vector also contains the gene sacB. This gene conferssensitivity to sucrose. Thus selection on sucrose can be used to detectisolates that have lost the plasmid and retain one copy of the deletionor wild-type genes ¹ S. elodea ATCC 31461 has a low efficiency of uptakeof DNA, particularly large plasmids (about 10⁻⁷). Spontaneous mutants ofATCC 31461 with increased efficiency of DNA uptake were isolated. It wassuspected that those few cells that were successful recipients of aplasmid such as the broad-host-range plasmid pLAFR3 represented mutantsin the recipient population with an increased ability to take up thisplasmid DNA. To allow loss of the plasmid, three transconjugantscontaining pLAFR3 were grown under nonselective conditions (i.e.,without tetracycline antibiotic) with serial passage for about 30generations. Three independent plasmid-cured strains (i.e.,tetracycline-sensitive derivatives from each of the initialtransconjugants) were tested and all three exhibited increasedconjugation frequency (4.2×10⁻³, 0.6×10⁻², and 1.5×10⁻²), representing a10⁵-fold increase compared to the wild-type strain. This increasedconjugation frequency was stable and reproducible. One of these strainswas designated S60 wtc. See reference 3.

A plasmid containing the gelM-gelN deleted region was introduced intoS60 wtc by tri-parental conjugal mating, using pRK2013 to providetransfer functions, and transconjugants selected on YM-Sm (25 ug/ml)-Km(7.5 ug/ml) medium. Streptomycin prevents growth of the E. coli strains.Kanamycin resistant plasmid integrants were isolated. Sucrosesensitivity was used to select for a second recombination event whicheliminated the vector. Five isolates were passed two times undernon-selective conditions, i.e., without antibiotic. Aliquots were thenplated on medium with 8% sucrose. Sucrose resistant colonies wereisolated and tested for kanamycin sensitivity. Genomic DNA was isolatedand PCR was used to determine which Km^(S) isolates had retained thedeletion. An amplified fragment of the expected size for a deletionresulted from the genomic DNA from four strains. These four deletionstrains were purified on YM medium. All four strains appeared lessmucoid, softer, flatter and darker yellow than the wild type.

EXAMPLE 2 Characterization of gelM-gelN Deletion Strains

The gelM-gelN deletion isolates were evaluated in shake flaskfermentations. The ΔgelM-gelN culture broth was fluid and smoothcompared to the more solid, viscous S60 wtc broth. Precipitation withisopropyl alcohol produced longer, thicker, fibers from the mutantstrains compared to S60 wtc fibers. However, the deletion mutants had22% reduction in yield of total precipitable material and produced only30% of the broth viscosity of wild-type. The gellan produced had anormal composition of sugars, and glyceryl and acetyl substituents.

The mutants were evaluated for slime forming characteristics, usingseveral techniques, including microscopic evaluation, cell clumping,cell pellet formation, and hot settling test, as shown in FIG. 2.

The hot settling test consisted of heating the gellan broth in theautoclave for ten minutes to melt the gellan, then transferring the hotbroth to a large test tube and incubating overnight at 95° C. (tomaintain broth as liquid). With a capsular strain, the cells areattached to the polysaccharide and remain suspended. For slime-formers,the cells are not attached and settle during overnight incubation. ThegelM-gelN deletion strains were shown to be slime formers by this test.

For the centrifugation test, the strains were grown overnight in DM2media containing 1% glucose and centrifuged at maximum speed in theEppendorf centrifuge. Inactivation of gelM-N genes results in completeloss of attachment of the polysaccharide from the cell surface such thatthe cells can be pelleted by centrifugation.

By microscopic evaluation, most of the S60 wtcΔgelM-N cells are free andmotile, whereas the S60 wtc are in the gum matrix. In cell culture, theS60 wtcΔgelM-N cells grow in suspension whereas S60 wtc cells formclumps.

EXAMPLE 3 Construction of Gellan Slime-Forming Mutants

A deletion was constructed of gelN for gellan biosynthesis. PCR primerswere designed to amplify DNA fragments upstream (500 bp) and downstream(401 bp) of the gelN gene [3]. Primers used are shown in Table 1.

TABLE 1 Primers for construction of gelN deletion mutant. PrimerSequence Purpose SacI-GelN primer 1 5′ TGGAGCTC-GGTGCTGTGGTTGTTCTT 3′Amplifies 500 bp upstream (SEQ ID NO: 1) of gelN XbaI-GelN primer 2 5′GGTCTAGA-GTCAGGCCGGTTGAACAT 3′ (SEQ ID NO: 2) XbaI-GelN primer 3 5′AGTCTAGA-GCCTGAACGCCGAAAGGG 3′ Amplifies 401 bp (SEQ ID NO: 3)downstream of gelN SphI-GelN primer 4 5′ GTTGCATGC-GGTGATGGTGGAGAATGG 3′(SEQ ID NO: 4)

Primers SacI-GelNprimer1 and XbaI-GelNprimer2 were used to amplify a 500bp fragment from the gelM gene as a SacI-XbaI fragment (total 516 bp).Primers XbaI-GelNprimer3 and SphI-GelNprimer4 were used to amplify a 401bp fragment from the atrD gene as an XbaI-SphI fragment (total 418 bp).Since the end of the gelM gene overlaps the start of the gelN gene by 17bp, the stop codon of gelM and the start codon of gelN were retained, aswell as the natural stop codon of gelN. The PCR fragments were ligatedsequentially into the polylinker of plasmid vector pLO2 [4], resultingin clone pLO2-gelNdeln#1 carrying the deletion of gelN.

Plasmid pLO2-gelNdeln#1 was then used to transfer the deletion to strainS60 wtc³ by conjugation and homologous recombination. Strain S60 wtc isa strain derived from ATCC 31461 as a spontaneous mutant with increasedability to take up plasmid DNA [2]. A chromosomal integrant was selectedby kanamycin resistance. Subsequent growth for approximately 30generations in the absence of antibiotic allowed for excision of theplasmid. Recombinants that had lost the plasmid were then selected bysucrose (8%) tolerance, due to loss of the plasmid-encoded sacB gene,and then colonies were screened for kanamycin sensitivity. The sacB geneencodes an enzyme levansucrase for synthesis of levan from sucrose.Levan is toxic to the cells. Cells that have lost the sacB gene can growon sucrose. The sucrose tolerant isolates can be wild-type or deletion.Genomic DNA was prepared from several isolates to identify thoseisolates that had retained the gelN deletion versus the wild-type gene,as determined by PCR. Isolates with the gelN deletion had softer, morewatery colonies compared to the hard colonies of the wild-type gelN+isolates. ³ See footnote 1 regarding use of mutant with increasedability to take up DNA.

EXAMPLE 4 Characterization of gelN Deletion Mutants

The gelN deletion mutants had similar properties to the gelM-gelNdeletion mutants. Cells were readily pelleted by centrifugation. In cellculture, the gelN deletion mutants grew in suspension, whereas thewild-type cells formed clumps. Thus, inactivation of the gelN gene canresult in the slime phenotype as shown in FIG. 3.

Five individual isolates of gelN deletion mutants were evaluated inshake flask fermentations. The average yield (total precipitablematerial, TPM) for the gelN mutants (1.10 g/100 ml) was comparable tothat of the S60 wtc control (1.08 g/100 ml). Selected gelN mutants wereevaluated in 20L Applikon fermentors using media containing organic andinorganic nitrogen, salts and corn syrup. Gellan polysaccharide wasprecipitated with isopropyl alcohol, dried and weighed. Average yield oftotal precipitable material for the mutants was 94% of that of thewild-type control, however the broth viscosity was decreased by about40%. This decrease in broth viscosity facilitates mixing in thefermentors.

TABLE 2 Fermentation characteristics of gelN mutants TPM Broth ViscosityStrain g/L Cp Wild-type 12.80 5500 gelN #1 12.04 3850 gelN #2 12.15 2700gelN #4 11.80 3400 average 12.00 3317Viscosity was measured in the Brookfield LVF viscometer using the No. 4spindle at 60 rpm.

EXAMPLE 5 Construction of Mutants Producing Gellan Slime with ImprovedQuality

The slime mutants of gellan had lower broth viscosity, as described inExample 4, which facilitates mixing in the fermentors. Gellanpolysaccharide forms a gel after heating and cooling, and gellan is usedin various food applications due to its unique textural and rheologicalproperties. Therefore, the gel strength of the gellan produced by theslime mutants was evaluated. The gel strength is determined by the breakor fracture of a prepared gel surface with a plunger.

The gellan fermentation broth was adjusted to pH 4.6 (to preventdeacylation) and pasteurized by heating to about 100° C. for severalminutes. Gellan product was precipitated by addition of three timesvolume of isopropyl alcohol, and the fibers dried at 60° C. for twohours and milled.

A calcium solution was prepared by adding 2 ml of a 0.3 M CaCl₂.2H₂Ostock solution to 295 ml of deionized water in a tared 600 ml stainlesssteel beaker. While stirring the solution at 700 rpm, 3.0 g of gellanproduct was added and allowed to disperse for 1 to 2 minutes. The beakerwas placed into a preheated water bath at 94-95° C. for four minutes,covered and heated for 15 minutes, then stirred for 3 minutes. Solutionweight was adjusted to 300 g with heated deionized water, mixed and thenleft standing at 94-95° C. Solution was transferred into six ring molds(0.5 inch height, 1.375 inch outer diameter, 0.125 inch wall thickness)and covered with a plastic cover plate and allowed to cool at roomtemperature (20-21° C.) for 20 to 24 hours. The disc was removed fromthe mold onto a plexiglass plate. Gel strength, or force to break,(recorded in g/cm 2) was determined in a TA-TX2 Texture Analyzer with aKobe plunger (TA-19) at 1.0 mm/s.

Gellan from the slime mutants had lower gel strength than that from thewild-type capsular stain, as shown in Table 4. This result was incontrast to mutants of ATCC 53159 that produce the slime form of diutan,which had improved rheology as described in Example 11. It wasconsidered possible that the slime form of gellan may be degraded by agellan lyase enzyme, produced by S. elodea. Therefore, a strain wasconstructed that has a deletion of a gene, gelR, which produces aprotein with homology to polysaccharide degrading proteins, e.g. lyases.

The gelR deletion was constructed in S. elodea strain S60 wtc and strainGBAD-1 (U.S. Patent application No. 20060003051 [11]). PCR primers weredesigned to amplify DNA fragments upstream (502 bp) and downstream (435bp) of the gelR gene. PCR primers used are shown in Table 3.

TABLE 3 Primers used for construction of gelR deletion Primer SequencePurpose SacI-GelR primer 1 5′ ACGAGCTCAGATCAGCCGCAACCTCCT 3′ Amplifies486 bp (Seq ID No :21) upstream of gelR XbaI-GelR primer 2 5′GCTCTAGA-CGCCGCCATGTTAATCACC 3′ (Seq ID No: 22) XbaI-GelR primer 3 5′GCTCTAGA-GATGCGTTCCACGCCTGAC 3′ Amplifies 419 bp (Seq ID No: 23)downstream of gelR SphI-GelR primer 4 5′ ATGCATGC-CGATCGCGCTCATCAGGGT 3′(Seq ID No: 24)

Primers SacI-GelR primer 1 and XbaI-GelR primer 2 were used to amplify a498 bp fragment upstream of gelR as a SacI-XbaI fragment (total 502 bp).Primers XbaI-GelR primer 3 and SphI-GelR primer 4 were used to amplify a419 bp fragment downstream of gelR as an XbaI-SphI fragment (total 435bp). The PCR fragments were digested with restriction enzymes andligated sequentially into the polylinker of plasmid vector pLO2 [4]resulting in clone pLO2-gelRdeletn #4, carrying the deletion of gelR.

Plasmid pLO2-gelRdeletn #4, which cannot replicate in Sphingomonas, wastransferred into S. elodea strains S60 wtc and GBAD-1 by conjugationfrom E. coli DH5α, using helper plasmid pRK2013 that supplies transferfunctions [2]. Chromosomal integrants were selected by kanamycinresistance on YM medium with kanamycin and streptomycin (tocounterselect E. coli). Subsequent growth of the Sphingomonas integrantsfor approximately 30 generations in the absence of antibiotic allowedfor the excision of the plasmid. Recombinants that had lost the plasmidwere selected by sucrose tolerance due to loss of the plasmid encodedsacB gene, and colonies screened for kanamycin sensitivity. PCR was usedto test which isolates had retained the gelR deletion.

The gelN deletion was than transferred into the gelR deletion mutant ofthe GBAD-1 strain as described above in Example 3. PlasmidpLO2-gelNdeln#1 was used to transfer the gelN deletion into GBADgelR byconjugation and homologous recombination. A chromosomal integrant wasselected by kanamycin resistance on YM agar with Km (20 ug/ml) and Sm(25 ug/ml). Subsequent growth in the absence of antibiotic allowed forexcision of the plasmid. Recombinants that had lost the plasmid wereselected by sucrose tolerance due to loss of the plasmid-encoded sacBgene and then colonies were screened for kanamycin sensitivity.

The gelR deletion mutants exhibited different colony morphology than thewild-type strains. The gelR deletion strains had smaller rough gummycolonies compared to larger smooth gummy colonies with transparent edgesfor the gelR+ of S60 wtc or GBAD-1. The gelN-gelR deletion mutants hadcolony morphology similar to the gelN slime mutants.

EXAMPLE 6 Characterization of gelN-gelR Mutants

These strains were evaluated in 20L Applikon fermentors using mediacontaining organic and inorganic nitrogen, salts and corn syrup. Gellanpolysaccharide was precipitated with isopropyl alcohol, dried andweighed. Gel strength was determined by the method described in Example5. The GBAD-1gelNgelR strain produced gellan of higher gel strength thanthe gellan produced from the gelN slime mutants or the wild-typecapsular strains.

TABLE 4 Rheological characterization of gellan from mutants Aver TPMAver. % of Broth Visc Aver Strain Phenotype n = wild-type cP Gelstrength S60wtc capsule 3 — 7292 411 GBAD1 capsule 1 91 4600 629 GelNmutants slime 7 93 2900 132 GBAD1gelRgelN slime 3 96 5083 1447

EXAMPLE 7 Construction of gelI Mutant

An insertion mutation in gene gelI was constructed. PCR primers weredesigned to amplify an internal fragment of the gelI gene (see reference3). The amplified fragment was cloned into the pLO2 plasmid vector andintroduced into S60 wtc by conjugation, selecting on YM-Sm (25 ug/ml)-Km(7.5 ug/ml) medium. Selection for kanamycin resistance selects for thosetransconjugants that have the plasmid inserted by homologousrecombination into the gelI gene, thus inactivating this gene. The gelImutant had altered colony morphology, similar to that of the gelM-gelNand the gelN deletion strains, i.e. mucoid but softer colonies.

EXAMPLE 8 Characterization of gelI Mutant Strain

The gelI mutant was evaluated in shake flask fermentation. The mutanthad less viscous broth compared to the wild-type strain and about a 20%reduction in yield of total precipitable material. The gellan producedhad a normal composition of sugars and glyceryl and acetyl substituents(reference 3).

The gelI mutant was evaluated for slime forming characteristic usingseveral techniques including microscopic evaluation, cell clumping, cellpellet formation and hot settling test as described above. The gelIinsertion mutant had similar characteristics to the gelM-gelN and thegelN deletion mutants. Microscopic evaluation showed that the cells werefree and motile. In cell culture, the gelI mutants grew in suspensionrather than clumps. Cells were readily pelleted from DM2 medium bycentrifugation. Cells also settled well in the hot settling test. Thus,the mutation in the gelI gene also results in the slime phenotype asshown in FIG. 3.

EXAMPLE 9 Production of Diutan Slime-Forming Mutants

Sphingomonas sp. ATCC 53159 (S-657) produces a polysaccharide (diutan)with a structure similar to that of gellan (i.e., it has aglucose-glucuronic acid-glucose-rhamnose repeat unit), but with a sidechain of two rhamnose residues attached to one glucose residue. Diutanhas two acetyl substituents, and lacks glyceryl groups. Diutan is usefulas a viscosifier in oilfield and cement applications. Sphingomonasstrains produce polysaccharides as capsules firmly bound to the cellsurface. The exact mechanism of attachment is not known. The capsule maylimit productivity by impairing oxygen uptake. The functionality of thepolysaccharide may be hindered by its being attached to the cell ratherthan free in solution.

Strain Construction:

We constructed deletions of the corresponding genes dpsM and dpsN ofSphingomonas sp. ATCC 53159, which produces diutan (S-657). Each genewas deleted independently and the effect on capsule to slime determined.Briefly, PCR was used to amplify two fragments homologous to DNAflanking the target gene. These fragments were cloned into anarrow-host-range plasmid pLO2 that cannot replicate in Sphingomonas andcontains two selective markers, kan^(R) and sacB. Selection forkanamycin resistance selects for cells in which the plasmid hasintegrated into the chromosome in one of the homologous regions. Thekanamycin resistant strain was then grown under nonselective conditionsto allow loss of the plasmid by a second recombination. Loss of plasmidwas selected by tolerance to sucrose. The sacB gene encodes an enzymelevansucrase for synthesis of levan from sucrose. Levan is toxic to thecells. Cells that have lost the sacB gene can grow on sucrose. Thesucrose tolerant isolates can be wild-type or deletion. Presence of thedeletion was confirmed by PCR. Mutants were tested for slime or capsuleproduction. No foreign DNA, plasmid, or antibiotic resistance genesremained in the final strain.

Detailed Construction of dpsN and dpsM Deletions Strains

Deletions of dpsM and dpsN were constructed on a plasmid and transferredto the genome of ATCC 53159, using a gene replacement strategy similarto that described for S. elodea deletion mutants [3]. PCR was used toamplify DNA regions flanking the target gene and then the fragmentscloned into plasmid pLO2 [4], which was then used to exchange thedeletion for the target gene in the chromosome. Primers used for the PCRare shown in Table 5. Restriction sites for cloning (shown in italics)were added to the ends of the primers.

TABLE 5 Primers for construction of deletion mutations. Primer SequencePurpose SacI-DpsN primer 1 5′ TTGAGCTC-GCTGTGGCTGTTCTTCCT 3′ Amplifies497 bp (SEQ ID NO: 5) upstream of dpsN XbaI-DpsN primer 2 5′CGTCTAGA-GTCACGCCGGTTGAACAT 3′ (SEQ ID NO: 6) XbaI-DpsN primer 3 5′TCTCTAGA-CTCGGTCACCAGGTCTGAA 3′ Amplifies 396 bp (SEQ ID NO: 7)downstream of dpsN SphI-DpsN primer 4 5′ CTCGCATGC-CGGTAAAGGTGAAG 3′(SEQ ID NO: 8) SacI-DpsM primer 1 5′ TTGAGCTC-GATCGGCGTTAAGACTGC 3′Amplifies 474 bp (SEQ ID NO: 9) upstream of dpsM XbaI-DpsM primer 2 5′CGTCTAGA-TCATCGCGGTCGCTGCCAT 3′ (SEQ ID NO: 10) XbaI-DpsM primer 3 5′CCTCTAGA-CGTCGGAGGCATCATGTTC 3′ Amplifies 509 bp (SEQ ID NO: 11)downstream of dpsM SphI-DpsM primer 4 5′ TCGCATGC-TCTGCTGATTGCCGTTCT 3′(SEQ ID NO: 12)

Deletion constructions were designed to leave the remaining genes fordiutan synthesis intact. For the dpsN deletion, primers SacI-DpsNprimer1and XbaI-DpsNprimer2 were used to amplify a 497 bp fragment from thedpsM gene as a SacI-XbaI fragment (total 513 bp). PrimersXbaI-DpsNprimer3 and SphI-DpsNprimer4 were used to amplify a 396 bpfragment from the atrD gene as an XbaI-SphI fragment (total 413 bp).Since the end of the dpsM gene overlaps the start of the dpsN gene by 17bp, the stop codon of dpsM and the start codon of dpsN were retained, aswell as the natural stop codon of dpsN. Thus this construction mayresult in formation of a small peptide of 13 amino acids, as shown inFIG. 2. The PCR fragments were ligated sequentially into the polylinkerof plasmid vector pLO2 [4], resulting in clone pLO2-dpsNdeln#3 carryingthe deletion of dpsN.

This plasmid, pLO2-dpsNdeln#3 which cannot replicate in Sphingomonas,was transferred into the Sphingomonas strain ATCC 53159 by conjugationfrom E. coli DH5α using a helper plasmid pRK2013 that supplies transferfunctions [2]. Chromosomal integrants were selected by kanamycinresistance on YM medium with 7.5 ug/ml kanamycin, and 25 ug/mlstreptomycin (to counterselect E. coli). Subsequent growth of theSphingomonas strains for approximately 30 generations in the absence ofantibiotic allowed for excision of the plasmid. Recombinants that hadlost the plasmid were then selected by sucrose (8%) tolerance, due toloss of the plasmid-encoded sacB gene, and then colonies screened forkanamycin sensitivity. Genomic DNA was prepared from several isolates toidentify those isolates that had retained the dpsN deletion versus thewild-type gene, as determined by PCR.

Similarly a deletion of dpsM was constructed. Primers SacI-DpsMprimer1and XbaI-DpsMprimer2 were used to amplify a 474 bp fragment from thedpsE gene as a SacI-XbaI fragment (total 490 bp). PrimersXbaI-DpsMprimer3 and SphI-DpsMprimer 4 were used to amplify a 509 bpfragment from the dpsN gene as a XbaI-SphI fragment (total 525 bp).Since the end of the dpsM gene overlaps the start of the dpsN gene by 17bp, the stop codon of dpsM and the start codon of dpsN were retained. Astop codon was incorporated within the XbaI cloning site. A 7-amino acidpeptide may be formed from the dpsM start site. The PCR fragments wereligated sequentially into the polylinker of plasmid vector pLO2 [4],resulting in clone pLO2-dpsMdeln#1 carrying the deletion of dpsM. Thisplasmid was transferred by conjugation into ATCC 53159 selecting forkanamycin resistant integrants, followed by growth in the absence ofantibiotic and detection of sucrose tolerant, kanamycin sensitiverecombinants. Genomic DNA was isolated from selected recombinants andscreened by PCR for presence of the deletion.

EXAMPLE 10 Characterization of Diutan Slime-Forming Mutants

Results of several tests showed that both the dpsM and dpsN deletionsresult in a change from capsule former to slime former, as shown inFIGS. 3 and 4.

-   -   1. Microscopic evaluation of two dpsN deletion mutants (#3 and        #5) and two dpsM deletion mutants (#1 and #5) grown about 16        hours in high carbon fermentation medium indicated that cells        from these mutants did not form the large cell aggregates        characteristic of the Sphingomonas capsular strain, S-657 (FIG.        3 A, and FIG. 4A).    -   2. Wild-type ATCC 53159 cells grown in defined medium (DM2) with        1% glucose for 24 hours and diluted ten-fold formed visible        clumps, where as the dpsM and dpsN slime mutants form uniform        suspensions similar to that of a non-mucoid strain, DPS1 (FIG.        3C for dpsN).    -   3. Centrifugation of 24-hour cultures grown in DM2 medium with        1% glucose showed that the cells from the dpsM and dpsN slime        mutants could be pelleted, whereas those from wild-type ATCC        53159 (S-657) remained attached to the polysaccharide and thus        did not pellet (FIG. 3B and FIG. 4B).

Six independent isolates of dpsN deletion mutants exhibited an average5.4% increase in total precipitable material compared to the wild-typecontrol, in shake flask fermentations. Selected dpsN and dpsM mutantisolates were evaluated in 20L Applikon fermentors using mediacontaining organic and inorganic nitrogen, salts and different carbonconcentrations (3-5%). Polysaccharide was precipitated with isopropylalcohol, dried and weighed. The dpsN mutants consistently exhibited aslight increase in total precipitable material compared to the wild-typecapsular control strain. The dpsM mutants gave more variable andgenerally lower productivity as shown in Table 6.

TABLE 6 Increase in yield of polysaccharide with dps mutants 5% carbonsource dpsN #3 n = 3 5.9% dpsN #5 n = 2 3.9% dpsM #1 n = 1 −30.2% dpsM#5 n = 1 9.3% 3% carbon source dpsN #3 n = 2 4.2% dpsM #1 n = 2 −10.1%dpsM #5 n = 4 −2.7%

EXAMPLE 11 Characterization of Diutan Slime-Form Polysaccharide

Rheological properties of diutan recovered from these fermentations byprecipitation with isopropyl alcohol was determined as shown in Table 7.Both dpsM and dpsN slime mutations resulted in improved viscosity ofdiutan.

TABLE 7 Rheological properties of diutan from slime mutants % 0.06 s−1 %% Strain SWV3 increase viscosity increase LSRV Increase wild- n = 5 26.727,760 2010 type dpsN n = 5 35.3 32% 37,920 37% 3873 93% #3 dpsN n = 237.3 40% 41,400 49% 4075 103%  #5 dpsM n = 3 40.5 52% 37,733 36% 390594% #1 dpsM n = 5 39.1 46%  39440 42% 3720 85% #5 aver. 42% aver. 41%aver. 94%

It was also observed that fiber quality, e.g., length, was improved withthe slime mutants. Since the polysaccharide molecules are free insolution rather than attached to the surface of the cell, theprecipitation of these molecules may be facilitated.

Low Shear Rate Viscosity Measurement.

Low shear rate viscosity is the viscosity of a 0.25% solution of diutanat 3 rpm. Standard or synthetic tap water was prepared by dissolving 10g of NaCl and 1.47 g of CaCl₂.2H₂O in 10 liters of deionized water. 4.5g of Polyethylene Glycol (PEG) 200 was weighed directly in a 400-ml tallform beaker. A 0.75 g aliquot of diutan product was weighed, anddispersed in the PEG 200 to form a consistent slurry. 299 ml ofsynthetic tap water was added to the beaker and the mixture stirred at800±20 rpm for approximately 4 hours. The beaker was removed from thestirring bench and placed in a 25° C. water bath and allowed to standfor 30 min. The viscosity was measured using a Brookfield LV Viscometerwith the No. 2 spindle at 3 rpm.

Seawater Viscosity Measurement.

Seawater viscosity was determined using the following procedure.Seawater solution was prepared by dissolving 41.95 g of sea salt (ASTMD-1141-52, from Lake Products Co., Inc. Maryland Heights, Mo.) per 980 gdeionized water, with pH adjusted to 8.2 with HCl or NaOH as needed. 307g of seawater solution was transferred to a mixing cup; 0.86 g of diutanproduct was slowly added over 15-30 seconds to the mixing cup andallowed to mix at 11,500 rpm for 45 minutes in the Fann Multi-Mixer,Model 9B5 (Fann Instruments, Inc, Houston Tex.). Three drops of BaraDefoam (NL Baroid/NL Industries, Inc., Houston, Tex.) was added andstirring was continued for an additional 30 seconds. The mixing cup wasremoved from the mixer and immersed in chilled water to lower thefluid's temperature, then placed in a constant temperature bath at 25°C. The solution was transferred to a 400-ml tall form beaker.

Fann viscosity (Fann Viscometer, Model 35A) was measured while mixing atlow speed (3 rpm). The shear stress value was read from the dial andrecorded as the SWV value at 3 rpm.

The viscosity was also determined on the Brookfield LV DV-II or DV-IIviscometer with the LV-2C spindle. The 0.06 sec⁻¹ reading was measuredat 0.3 rpm.

EXAMPLE 12 Materials and Methods

Medium. YM contains per liter, 3 g yeast extract, 5 g peptone, 3 g maltextract, and 10 g glucose. DM2 medium contains per liter, 2.68 g K₂HPO₄,1.31 g KH₂PO₄, 2.0 g NH₄SO₄, 0.1 g MgSO₄.7H₂O, 15 mg CaCl₂.2H₂O, 8.34 mgFeSO₄.7H₂O, 0.05 mg MnCl₂.4H₂O, 0.03 mg CoCl₂.6H₂O, 0.8 mg CuSO₄.5H₂O,0.02 mg Na₂MoO₄.2H₂O, 1.0 mg ZnSO₄.7H₂O, 0.2 mg H₃BO₃ and 10 g glucose.Gellan shake flask fermentation medium contains per liter, 0.23 g NaCl,0.165 g CaCl₂.2H₂O, 2.8 g K₂HPO₄, 1.2 g KH₂PO₄, 1.9 g NaNO₃, 1.0 gN-Z-Amine type EKC (Sheffield Products), 36.46 g Star-Dri corn syrup,2.5 mg FeSO₄.7H₂O, 24 μg CoCl₂.6H₂O and 0.1 g MgSO₄.7H₂O.

Centrifugation test for slime. Strains were grown approximately 24 hoursat 30° C. in DM2 medium containing 1% glucose, with shaking at 350 rpmand then centrifuged at maximum speed (10,000 rpm) for 5 minutes in theEppendorf centrifuge.

Hot settling test. Strains were grown in gellan shake flask fermentationmedium. Fermentation broth was heated in the autoclave for 10 minutes toliquefy gellan. The hot broth was then transferred to a large test tubeand allowed to settle overnight at 95° C. (to maintain broth as liquid).With a capsular strain the cells are attached to the polysaccharide andremain suspended. For slime-formers, the cells are not attached andprecipitate during overnight incubation.

PCR amplification. We used the high fidelity PCR enzyme “PfuUltra hotstart DNA polymerase” from Stratagene (LaJolla, Calif.).

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   1. Coleman R C. 2001. Cloning and analysis of Sphingomonas sp. ATCC    53159 polysaccharide genes. San Diego State University MS thesis-   2. Ditta G, S Stanfield, D Corbin and D R Helinski. 1980. Broad host    range DNA cloning system for Gram-negative bacteria: construction of    a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:    7347-7351.-   3. Harding N E, Y N Patel and R J Coleman. 2004. Organization of    genes required for gellan polysaccharide biosynthesis in    Sphingomonas elodea ATCC 31461. J Ind Microbiol Biotechnol 31:70-82.-   4. Lenz, O., E. Schwartz, J. Demedde, M. Eitinger and B.    Friedrich. 1994. The Alcaligenes eutrophus H116 hoxX gene    participates in hydrogenase regulation. J. Bacteriol. 176:4385-4393.-   5. Matthews T D. 2004. Identification of genes involved in    phenotypic phase shifting of Sphingomonas sp. ATCC 53159 San Diego    State University MS thesis-   6. Pollock T J and R W Armentrout. 1999. Planktonic/sessile    dimorphism of polysaccharide-encapsulated Sphingomonads. J Ind    Microbiol Biotechnol 23: 436-441.-   7. Pollock, T J, W A T van Workum, L Thome, M Mikolajczak, M    Yamazaki, J W Kijne and R W Armentrout. 1998. Assignment of    biochemical functions to glycosyl transferase genes which are    essential for biosynthesis of exopolysaccharides in Sphingomonas    strain S88 and Rhizobium leguminosarum. J Bacteriol 180: 586-593.-   8. Sa-Correia I, A M Fialho, P Videira, L M Moreira, A R Marques and    H Albano. 2002. Gellan gum biosynthesis in Sphingomonas paucimobilis    ATCC 31461: Genes, enzymes and exopolysaccharide production    engineering. J Ind Microbiol Biotechnol. 29: 170-176.-   9. Yamazaki M, L Thome, M Mikolajczak, R W Armentrout and T J    Pollock. 1996. Linkage of genes essential for synthesis of a    polysaccharide capsule in Sphingomonas strain S88. J Bacteriol 178:    2676-2687.-   10. Yamazaki M, M Mikolajczak, T J Pollock and R W Armentrout. 2003.    Production of exopolysaccharides unattached to the surface of    bacterial cells. U.S. Pat. No. 6,605,461.-   11. Cleary, Joseph M., et al. Genetically purified gellan gum. U.S.    Patent Application No. 20060003051.

1. A composition comprising a diutan polysaccharide which is produced by a process comprising recovering a diutan from a mutant Sphingomonas strain which contains at least one genetic modification that favors the production of diutan in a slime form, wherein said diutan as naturally produced by said mutant Sphingomonas strain imparts to a fluid an increased viscosity relative to an equivalent amount of diutan as naturally produced by a wild-type Sphingomonas strain ATCC 53159 wherein said gene mutations or deletions are in one or both of dpsN and dpsM genes and the Sphingomonas strain has deletion mutations in either or both dpsN and dpsM genes.
 2. The composition of claim 1 wherein the increased viscosity is at least 30% more than an equivalent amount of diutan produced by strain ATCC
 53159. 3. The composition of claim 1 wherein the increased viscosity is at least 40% more than an equivalent amount of diutan produced by strain ATCC
 53159. 4. The composition of claim 1 wherein the increased viscosity is at least 50% more than an equivalent amount of diutan produced by strain ATCC
 53159. 5. The composition of claim 1 wherein the increased viscosity is at least 60% more than the relative to an equivalent amount of diutan produced by strain ATCC
 53159. 6. The composition of claim 1 wherein the increased viscosity is at least 70% more than the relative to an equivalent amount of diutan produced by strain ATCC
 53159. 7. The composition of claim 1 wherein the increased viscosity is at least 80% more than the relative to an equivalent amount of diutan produced by strain ATCC
 53159. 8. The composition of claim 1 wherein the increased viscosity is at least 90% more than the relative to an equivalent amount of diutan produced by strain ATCC
 53159. 9. The composition of claim 1 wherein said mutant Sphingomonas strain is a slime forming Sphingomonas strain.
 10. The composition of claim 9 wherein said mutant Sphingomonas strain has one or more gene mutations or deletions which convert the strain from capsule former to a slime-forming mutant.
 11. The composition of claim 10 wherein the diutan produced by said mutant Sphingomonas strain comprises longer fibers than diutan as naturally produced by the wild-type Sphingomonas strain ATCC
 53159. 12. The composition of claim 1 wherein the diutan is produced by a variant of Sphingomonas strain ATCC 53159 wherein said variant has been genetically engineered to eliminate an expression of dpsN gene.
 13. The composition of claim 1 wherein the diutan is produced by a variant of Sphingomonas strain ATCC 53159 wherein said variant has been genetically engineered to eliminate an expression of dpsM gene.
 14. The composition of claim 1 wherein the diutan is produced by a variant of Sphingomonas strain ATCC 53159 wherein said variant has been genetically engineered to eliminate an expression of both dpsM and dpsN genes. 